The present invention relates to supports for immobilizing DNA or the like, particularly to supports which are chemically modified, more particularly to supports which are chemically modified by a hydroxyl group, a carboxyl group and so on at the terminal thereof.
In a conventional process, in order to obtain a specific amount of target DNA, the following heat cycle 1) to 3) has to be repeated in a DNA amplification reaction and so on:
1) the temperature of a test material is increased to 95xc2x0 C. in order to break the hydrogen bonds of the double chain;
2) the temperature of the test material is decreased to 45xc2x0 C. in order to reproduce DNA by adding a primer; and
3) the temperature of the test material is increased to 74xc2x0 C. in order to reproduce DNA by extending the primer with heat-resistant polymerase.
In such a DNA amplificaiton reaction, the test material is filled into a container made from synthetic resin. The container is installed in an aluminum block and heat-cycled.
However, it requires many hours to accomplish the heat cycle. It requires several hours to obtain a target amount of DNA. There is a drawback that others kinds of DNA are also reproduced in addition to the target DNA since the accuracy of thermal control is low.
To resolve the above drawback, a subject of the present invention is to provide a support suitable for immobilizing DNA easily and reproducing DNA by a DNA amplification reaction.
A support according to the present invention is comprises at least one component selected from the group of diamond, including non-diamond carbon, amorphous carbon, and graphite.
The support preferably has a chemical modification such as polar group, hydroxyl group or carboxyl group at its terminal.
The carboxyl group is preferably linked to a surface of the support through an ester linkage or a peptide linkage.
Regarding carbon material such as diamond including non-diamond carbon, amorphous carbon and graphite and so on, carbons are exposed on a surface of the support so that the surface can be chemically modified with a hydroxyl group, a carboxyl group and so on and DNA and so on can be immobilized easily. It is the most suitable to reproduce DNA and so on by a DNA amplification reaction.
Even if a surface of the support according to the present invention is contaminated, chemical modification can be reproduced by hydrolyzing.
The support according to the present invention comprises diamond including non-diamond carbon, amorphous carbon, graphite and so on. Although any methods for producing the surface can be used, a microwave plasma CVD method, an ECR CVD method, a high frequency plasma CVD method, an IPC method, a DC sputtering method, an ECR sputtering method, an ion plating method, an arc ion plating method, an EB evaporation method, a resistance heating evaporation method and so on are preferable. For example, carbon may be amorphous carbon with hydrogen obtained by steam-baking a resist layer made of polyimide material. Further, carbon may be sintered slurrying resin mixed with graphite powder. In the present invention, carbon may be selected from one or more than materials described above.
A surface of the support according to the present invention may be roughened. Such a rough surface has a relatively large surface area on which a large amount of DNA can be immobilized. The shape of the support may be a plate shape, a ball shape, polygon shape or various shapes. The material of the support may be mixed with the above described material and the other components. The support according to the present invention may be exposed on a surface of an object.
In the next, a specific group is chemically modified (added) on a surface of the above support. By providing a chemical modification, DNA is able to be immobilized on the surface of the support. A hydroxyl group, a carboxyl group, a surface group, a cyano group, a nitro group, a thiol group, an amino group and so on may be utilized as a specific group having a polar group and chemically modified on a surface of the support. In addition, an organic carbonic acid is also available.
The carboxyl group may be bonded to a terminal of the support through the other hydrocarbon group. In such a case, it is preferable that the number of carbons in the hydrocarbon group is from zero to 10 in order to immobilize DNA. Regarding the acid which can be charged to be a hydrocarbon group, a mono-carboxylic acid such as formic acid, acetic acid, propionic acid, di-carboxylic acid such as oxalic acid, malonic acid, succinic acid, maleinic acid, fumaric acid, phthalic acid and a polycarboxylic acid such as trimellitic acid are considerable.
In the case of utilizing the support according to the present invention for a DNA amplification reaction, there are two options, that is, one option is to require a hydrolysis-resist characteristic and another option is to require a reproduction of chemical modification by hydrolyzing.
In the case of requiring the hydrolysis-resist characteristic, it is preferable that a carboxyl group bonded to a terminal end of the hydro-carbon group is bonded to a surface of the support through a peptide linkage in order to provide alkali-proof characteristic.
On the other hand, in the case of requiring a reproduction of chemical modification by hydrolyzing and removing the produced chemical modification, it is preferable that a carboxyl group bonded to a terminal end of the hydro-carbon group is bonded to a surface of the support through an ester linkage.
Regarding a method for bonding a hydroxyl group to a terminal end of a hydro-carbon group on the surface of the support, a method for oxidizing a surface of the support with oxygen plasma and treating with steam, a method for chloridizing a surface of the support by irradiating ultra violet beam in chloride gas and hydrolyzing the support by hydrolyzing in alkali solution are suitable.
Methods for linking the carboxyl group bonded to the terminal end of the hydro-carbon group on the surface of the support through the peptide linkage, include chloridizing a surface of the support by irradiating with ultra violet radiation in chloride gas, aminatizing the support by irradiating with ultra violet radiation in ammonia gas, reacting with carbonic chloride in a non-water soluble solution and neutralizing in alkaline solution.
Methods for bonding the carboxyl group connected to a terminal end of the hydrocarbon group on a surface of the support through an ester linkage, include chloridizing the surface of the support by irradiating with ultra violet radiation in chloride gas, reacting with sodium carbonate in non-water soluble solution and neutralizing in a weak acid solution and a oxidizing a surface of the support with oxygen plasma, chloridizing, hydrolyzing by hydrolyzing in alkali solution, reacting with carbonic chloride in non-water soluble solution and neutralizing in alkaline solution.